Dynamic measurement of the calcium buffering properties of the sarcoplasmic reticulum in mouse skeletal muscle

Abstract

Key points• The rise in cytosolic calcium ion concentration that triggers muscle contraction requires release of a large amount of calcium from the cellular store, sarcoplasmic reticulum (SR), where it is stored bound, largely to the protein calsequestrin.• Binding of calcium by calsequestrin is a complex process, believed to involve changes in protein conformation and aggregation. We want to know to what extent these properties, observed in vitro, apply inside cells.• We measured the calcium buffering power of the SR, defined as the ratio of change in total SR calcium by change in free [Ca 2+ ] SR , in muscle cells of wild type or calsequestrin-lacking mice, using two different methods to monitor [Ca 2+ ] SR and deriving changes in total SR calcium content from simultaneous measurements of cytosolic [Ca 2+ ].• The average buffering power during a large, depleting calcium release event was 157 in the wild type and 40 in the calsequestrin-null mice, suggesting that three-quarters of the calcium released normally comes from the calsequestrin-bound pool.• The Ca 2+ buffering ability of the SR is different from that of the equivalent concentration of calsequestrin in aqueous solution, the SR exhibiting greater affinity and cooperativity. We conclude that calsequestrin adopts different properties inside cells.• SR buffering power depends on the SR Ca 2+ load and on the rate of its changes, a dependence that could be, at least in part, explained by the unique Ca 2+ binding properties of calsequestrin.• This study reveals Ca 2+ buffering as a highly dynamic process, marking it as both a vulnerable link in diseases that involve loss of control of Ca 2+ release, and a candidate for further study and intervention.Abstract The buffering power, B, of the sarcoplasmic reticulum (SR), ratio of the changes in total and free [Ca 2+ ], was determined in fast-twitch mouse muscle cells subjected to depleting membrane depolarization. Changes in total SR [Ca 2+ ] were measured integrating Ca 2+ release flux, determined with a cytosolic [Ca 2+ ] monitor. Free [Ca 2+ ] SR was measured using the cameleon D4cpv-Casq1. In 34 wild-type (WT) cells average B during the depolarization (ON phase) was 157 (SEM 26), implying that of 157 ions released, 156 were bound inside the SR. B was significantly greater when BAPTA, which increases release flux, was present in the cytosol. B was greater early in the pulse -when flux was greatest -than at its end, and greater in the ON than in the OFF. In 29 Casq1-null cells, B was 40 (3.6). The difference suggests that 75% of the releasable calcium is normally bound to calsequestrin. In the nulls the difference in B between ON and OFF was less than in the WT but still significant. This difference and the associated decay in B during the ON were not artifacts of a slow SR monitor, as they were also found in the WT when [Ca 2+ ] SR was tracked with the fast dye fluo-5N. The calcium buffering power, binding capacity and non-linear binding properties of the SR measured here could be accounted for ...

Calsequestrin depolymerizes when calcium is depleted in the sarcoplasmic reticulum of working muscle

“…Indeed, strongly cooperative calcium buffering by the SR was first documented by Pape et al (31), who showed with frog muscle that the calcium buffering power of the empty SR is negligible compared with the value at rest, a property they attributed to calsequestrin. Working on mouse muscle, Royer et al (19) confirmed the attribution, quantified the contribution by Casq1 at 75% of released calcium (26), and equated it to the hump of calcium flux caused by a large depolarization (19). Here, we characterized this component further (Fig.…”

“…S4, it increased markedly in the first bleached region of on Casq-null mice. Most, however, were done on WT mice; therefore, the exogenous Casq1 (which was expressed at 2-5 μmol/L cytosol) was less than 10% of the total [estimated at 50 μM or greater (25,26)]. The evolution of f ðtÞ in a representative case is shown in Fig.…”

“…Given that Casq1 provides much of the calcium that signals muscle contraction (25,26), the buffering features found in solution should transfer to the storage properties of the SR in cells. Indeed, strongly cooperative calcium buffering by the SR was first documented by Pape et al (31), who showed with frog muscle that the calcium buffering power of the empty SR is negligible compared with the value at rest, a property they attributed to calsequestrin.…”

Characterization of Two Human Skeletal Calsequestrin Mutants Implicated in Malignant Hyperthermia and Vacuolar Aggregate Myopathy

“…Calsequestrin (2) (protein and genes denoted here as Casq) is the most abundant and capacious Ca 2ϩ -binding protein within the SR of both skeletal and cardiac muscle where tissue-specific isoforms Casq1 and Casq2, respectively, are expressed. In skeletal muscle, ions bound to Casq1 constitute ϳ75% of the Ca 2ϩ released for activation of contraction (3). Ca 2ϩ titrations of Casq in vitro feature multiple stages associated with progressive Casq polymerization (4,5).…”

Leaky ryanodine receptors delay the activation of store overload-induced Ca2+ release, a mechanism underlying malignant hyperthermia-like events in dystrophic muscle

“…Shifts in the threshold for SOICR occur through chronic or acute modifications of the RyR (6,8,17). Importantly, the activation of propagating SOICR is associated with a depolymerization of calsequestrin (CSQ), the SR Ca 2ϩ buffer, and a subsequent reduction in the power of the SR to buffer Ca 2ϩ (B SR ) (8,25,27,31). Little is known of the activation of SOICR and associated changes in B SR in skeletal muscle under physiological conditions.…”